Abstract

Introduction: We recently demonstrated that transcription of the ribosomal genes (rDNA) by RNA Polymerase I (Pol I) can be therapeutically targeted with a novel small molecule, CX-5461, to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B cell population (Bywater et al Cancer Cell 2012; Bywater et al Nature Reviews Cancer 2013). The therapeutic effect was a consequence of nucleolar disruption and activation of ribosomal protein (Rp)-MDM2-P53 nucleolar stress response (3) leading to apoptosis. Human leukemia and lymphoma cell lines also show high sensitivity to inhibition of rDNA transcription that is dependent on p53 mutational status. As our pre-clinical data indicate immense potential of Pol I targeting as a cancer therapy, we have launched a first-in-human clinical trial of CX-5461 in patients with hematological malignancies. Despite impressive initial responses with CX-5461 as a single agent, we decided to explore its combination potential. We hypothesised that simultaneously targeting the ribosome at multiple steps would reduce the instance of acquired resistance to CX-5461 and extend survival. Thus we tested pharmacological inhibitors of the PI3K/AKT/mTOR pathway in combination with CX-5461 as the former signaling molecules are known to be potent regulators of both ribosome translational activity (Jefferies et al EMBO J, 1997; Pourdehnad M et al PNAS 2013) and ribosome biogenesis (Chan et al Science Signaling 2011; Devlin et al FEBS J 2013).

Experimental procedures and new data: Using the Eμ-Myc model of B-cell lymphoma we demonstrate that multiple pharmacological inhibitors of the PI3K/AKT/mTOR pathway also suppress transcription of the rRNA genes and induce cell death to a similar extent as CX-5461. Unexpectedly however, PI3K/AKT/mTOR pathway blockade is not associated with nucleolar disruption, nor activation of the Rp/MDM2/p53 nucleolar stress pathway. We demonstrate this is because inhibition of PI3K/AKT/mTOR suppresses both rRNA synthesis and ribosomal protein synthesis equally and therefore does not result in a pool of free Rps that are necessary to suppress MDM2 E3 ligase that regulates p53 stability. Instead apoptosis induced by PI3K/AKT/mTOR was associated with up-regulated expression of the pro-apoptotic BH3-only protein BMF. Furthermore, we demonstrate that combined treatment of Eμ-Myc tumour-bearing mice with CX-5461 and Everolimus delayed relapse compare to single agent and significantly extended survival of tumor bearing mice.

Conclusions: These data demonstrate that dual targeting of the ribosome by selectively inhibiting Pol I transcription and by inhibition of key signaling molecules regulating ribosome synthesis and function combine to potently treat MYC driven tumours and provides a rationale to combine such drugs in the clinic for the treatment of MYC driven cancer. Moreover these data also demonstrate that MYCs control of Pol I transcription and nucleolar integrity is required for its oncogenic potential, independent of its well described function in function in controlling ribosome levels and protein translation.